The invention relates to a method for production of a heterologous protein, for which host cells of a yeast type are prepared that contain in each case at least one recombinant DNA sequence coding for the heterologous protein, then the host cells are caused to express and secrete the heterologous protein, and the secreted, heterologous protein is separated.
Methods of the type mentioned in the introduction are known, for example, from publication WO 00/68400 A1. The obtaining of proteins, which, for example, are used as active compounds in medications, with the help of recombinant DNA technology, has, among others, the advantage that the proteins can be made available in well characterised hosts in practically unlimited quantities. It is advantageous for the obtaining of recombinant proteins if, in each case, the expressed protein is secreted so that it can subsequently be obtained from the cell supernatant. This greatly simplifies the preparation, because the protein is already available in a relatively pure form and costly purification steps can be avoided. In many cases, yeasts are used in order to obtain secreted proteins on a large scale. These have the advantage that they can be kept relatively simply in cell cultures and lead to a good yield.
It is always the aim to increase the secretion of the heterologous proteins. In order to achieve this, it was proposed, for example, to increase the number of copies of the recombinant DNA sequences that code for the heterologous protein. Further, for an increase in yield of the protein, usually secreted in glycosylated form, it was proposed by Arima et al in ‘Enhanced secretion of hydrophobic peptide fused lysozyme by the introduction of N-glycosylation signal and the disruption of calnexin gene in Saccharomyces cerevisiae,’ PEBS Letters 440 (1998), pages 89-92, to deactivate the gene of the yeast cells that code for the chaperone calnexin. Based on the results of this work, it can be expected that the calnexin reduces the secretion of heterologous proteins from yeast cells. In ‘Calnexin Overexpression Increases Manganese Peroxidase Production in Asperigillus niger,’ in Applied and Environment Microbiology, February 2002, pages 846-851, Conesa et al describe that an overexpression of calnexin increases the production of manganese peroxidase; however, on the one hand, this applied to filamentary fungus cultures, and, on the other hand, the positive influence of the calnexin overexpression on the yield was caused by the calnexin influencing heme incorporation into the manganese peroxidase.